Project Summary Parkinson's disease (PD) is a common neurodegenerative disorder characterized by dopaminergic neuron degeneration and the aggregation of misfolded proteins. Except for symptomatic relief, no therapy is currently available to prevent or reverse the disease process. One of the major obstacles for developing an effective treatment is the lack of methods to diagnose PD prior to the clinical symptoms, which usually occur after the loss of a large amount of dopaminergic neurons in the substantia nigra. The overall goal of our project is to develop a sensitive and definitive method for early diagnosis of PD. We are targeting the alpha-synuclein (?- syn) aggregates because they appeared early during the disease process and are intimately associated with the disease progression. Taking advantage of the seeding capability of ?-syn aggregates, we propose to use the protein misfolding cyclic amplification (PMCA) method to measure the capabilities of ?-syn aggregates to seed recombinant monomeric ?-syn to assemble into amyloid fibrils, which is able to significantly amplify the signal and allow us to detect a minuscule amount of ?-syn aggregates. In this application, we designed novel strategies in order to significantly improve the sensitivity and specificity of the assay, which allow us to overcome the major obstacles blocking the application of ?-syn PMCA in clinical settings. Although these are mainly technical improvements, if successful, they will transform ?-syn PMCA from a bench side experimental assay into a useful clinical test, which has the great potential to develop into an early diagnostic tool for PD or other synucleinopathies. Moreover, the target measured in our assay, ?-SN aggregates with seeding capability, also has the potential to develop into a useful biomarker for monitoring disease progression, estimating therapeutic efficacy, and/or stratifying this complicated neurodegenerative disease.